Hfq is a bacterial Sm-like protein involved in several aspects of RNA biochemistry. This protein facilitates RNA-RNA interactions during post-transcriptional gene regulation. In these pathways, small non-coding RNAs (ncRNAs) regulate mRNA targets to facilitate adaption to environmental conditions like cold shock, heat shock, osmotic shock and oxidative stress. Hfq interacts tightly with many RNA partners and yet it seems to assemble the correct ncRNA-mRNA partnerships with high fidelity without becoming trapped in inactive complexes. The work in this proposal seeks to understand from a chemical and biological perspective how Hfq functions. Aim 1 looks at the molecular handshakes involved in the strand displacement reaction which alters the translational state of the structurally repressed rpoS mRNA. We have evidence that this interaction involves ternary complexes and seek to trap and analyze intermediates along the reaction pathway. We will then test the validity of our models based on in vitro data by assessing in vivo, the effect of these mutations. Aim 2 probes more deeply the binary interactions invovled in Hfq's recognition of its ncRNA and mRNA partners. By using nucleotide analog interferences mapping and photocrosslinking studies we will characterize the structural features and atomic contacts required for high affinity binding. Aim 3 further analyzes a series of Hfq mutants that we have previously generated. Using this library of mutants, we have begun to differentiate the contact surfaces involved in Hfq's various biological functions. As a continuation of these studies in vivo, we will be addressing the question of whether additional protein components are involved in some of these pathways as part of a larger macromolecular assemblages. Aim 4 takes our work in a new direction. Hfq has recently been implicated in bacterial virulence in several organisms. We have now shown that a novel ncRNA called VR-RNA from C. perfingens (an organism that causes gas gangrene among other ailments) binds Hfq with high affinity. We will be map how Hfq and VR-RNA are invovled in virulence of this Gram positive organism and begin to understand the similarities and differences in these regulatory networks. [unreadable] [unreadable]